The objectives of this research proposal are to elucidate the cellular mechanism by which heat kills mammalian cells in culture and by which thermotolerance develops in these cells. The importance of this research is that the clinical use of hyperthermia can be optimized only when the basic biology of heat- induced cell killing and thermotolerance is better understood. The use of flow cytometry to measure cellular properties of individual cells in a large population will be utilized as a means toward this end. The changes in intracellular pH induced by hyperthermia at normal or low pH will be analyzed. The role of intracellular K+ and the Na+/H+ pump in modulating this effect will be investigated. The effects of low pH on cell cycle delays and heat shock protein synthesis will also be studied. Intracellular pH will be measured with the fluorescent dye, ADB, as well as DMO and NMR techniques. The role of intracellular calcium ion levels, and changes induced by hyperthermia, will be studied by fluorescence techniques, using Indo-1, and by NMR techniques. Cells will be sorted and plated for viability after fluorescent labeling for intracellular calcium, to correlate measurements with cell survival. The role of the plasma membrane fluidity changes in heat sensitivity will be studied with the dye, TMA-DPH (trimethyl- ammonium diphenyl hexatriene), by measuring the polarization of the dye in the membrane under conditions of thermotolerance. The lateral mobility of lipid in membranes will also be measured with fluorescence photobleaching recovery (FPR) techniques. Membranes potential will be measuring with oxacarbocyanine and oxonol dyes. The effects of the anesthetic, procaine, will also be determined by these techniques. Changes in cell volume and morphology induced by hyperthermia will be analyzed by using forward and 90 degree light scattering, and Coulter volume. Volume changes will be correlated with ionic changes.